future peptide vaccine for tb endemic regions: challenges ... · vaccine candidate with both...
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Pradeep K Rai CSIR-IMTECH, Chandigarh
Future peptide vaccine for TB endemic regions: Challenges and solutions
Epidemiology of Tuberculosis
Total no. of new TB cases 9 million
Previously treated 85%
New cases 15%
480,000
MDR: Multi drug resistant
WHO Global tuberculosis report , 2014
Total no. of deaths 1.5 million
Best Prophylactic measure
Vaccine
BCG
BCG Failure
Drugs
DOTs Treatment
Side effects
Emergence of drug resistant
Reasons for BCG failure
Lack of enduring immunologic memory (Andersen and Doherty 2005, Narayanan 2006, Orme 2010, Singh et al 2010, 2011)
Interference by environmental mycobacteria (Masking and Blocking hypotheses) (Andersen and Doherty 2005, Verma and Grover 2009, Flaherty et al 2006, Young et al 2007, Brant et al 2002)
Antigen processing and presentation- Blocking phagosome maturation and export of major histocompatibility complex (MHC) molecules (Soualhine et al 2007, Pancholi et al 1993, Sendide et al 2005, Sun et al 2007, Fulton et al 2004, Sendide et al 2004)
Helminth infections (Malhotra et al 1999, Elias et al 2001, 2005, Black et al 2002, Rook et al 2004)
Regulatory T cells (Hanekom 2005, Ahrens et al 2009, Rook and Kim 2006, Akkoc et al 2010)
Limitations of DOTs
Time consuming (3-12 months), so chances of intermittent therapy increases, resulting in increased risk of drug resistance. Side Effects. No cure for latent infection.
Urgent need to develop: A potent construct with two edge sword (Prophylactic and Therapeutic potential)
Future TB vaccine: Epitope based vaccine
Minimal antigen processing Minimal presence of mycobacterial components (except antigenic portions) Robust Th1 response (even in a Th2 bias environment) Enduring T cell memory No preformed antibodies in the population
Vaccine candidate with both prophylactic and therapeutic properties
Х Poor immunogenicity
Х Binds to very few HLA-alleles
HLA DRB1 Binding Affinity
F91-110
0101 ++++
0103 ++++
0301 ++
0401 ++++
0701 ++++
1101 ++++
1301 +++
1501 ++++
1601 ++++
++++ indicates high-affinity binding (IC50<10µM); +++ intermediate affinity (10 µM<IC50<100µM); and ++ indicates low affinity (100–1000µM).
Agrewala and Wilkinson 1997, 1998, 1999
Which peptide to choose? Is promiscuous peptide right choice?
Pam2Cys S-[2,3-is(palmitoyloxy)propyl]cysteine induces effective
Th1 immune responses, by evoking DCs to secrete IL-12 (Thoma et al 2000, Jackson et al 2004, Zho et al 2004, Ghosh et al 2006)
Dendritic cells copiously express TLR2 so Pam2Cys (TLR2 agonist)
can be used as vaccine delivery module (Jackson et al 2004, Zho et al 2004)
Pam2Cys has ability to mature and activate DCs (Hertz et al 2001, Jackson et al 2004, Zho et al 2004)
Safe for Human use (Zeng et al 2002)
-K I S I S I
Pam2Cys
SEFAYGSFVRTVSLPVGADE (91-110)
L91
Can coupling promiscuous peptides to Pam2Cys render them
immunogenic?
CD4
MΦ
Induction of MHC expression
Induction of microbicidal activity
NOS2 generation
Mechanism of L91
IFNγ , IL-17, TNFα
L91 confers better protection than BCG in Guinea pig model of tuberculosis
Placebo L91 BCG F91 LH
Whether L91 has therapeutic potential?
L91 efficiently decreases bacterial burden in the lungs and spleen in conjunction with anti-TB drugs
Lu
ng
s (
log
10
CF
U/g
)
*** ***
** ***
***
ns
*** ***
**
***
*** ns
Sp
leen
(lo
g1
0 C
FU
/g)
0 4 6 8 20 weeks
Mtb
infection
INH+RIF
L91 L91 CFU count
T cell response
L91 immunization reduces Mtb induced lung pathology
Placebo
L91
Drug
Drug-L91
20X 100X
L91 has enough therapeutic potential
to confine bacterial growth in lungs
Further, L91 restricts dissemination
of bacteria in spleen
Mechanism of protection?
iNOS
ACTIN
L91 stimulation elicits innate immunity
NF-kB
FB US F91 Pam L91 LPS
L91 immunization elicits secretion of protective cytokines against Mtb
3
4
5
6
7
8
9
PBS Drug L91 Drug+L91
IL-1
β (
S.I
) **
**
0
5
10
15
20
25
PBS Drug Drug+L91 L91
IL-1
7A
(S
.I)
***
**
0
5
10
15
20
25
30
PBS Drug L91 Drug+L91
IFNγ
(S.I
)
*** ***
ns
Lungs Single cell
suspension
In vitro stimulation
with L91
T cell response
72h
**
*
IFN
-γ+ T
NF
-α +
CD
4 T
cells (
%)
* *
IL-1
7+IF
N-γ
+ C
D4
T c
ell
s (
%)
L91 immunization induces generation of multifunctional CD4 T cells
*
*** C
XC
R3
+ C
CR
6+
IF
N-γ
+ IL
17
A+
CD
4 T
Ce
lls
(%
)
L91 immunization generates specific subtype of Th17 cells
PD
-1 (
iMF
I)
** *
**
*
ns
L91 immunization rescues CD4 T cells from exhaustion
CD
44
hi C
D62
Lh
i CD
4 T
ce
lls
(%
)
* **
**
**
CD
127
+ C
D4 T
cells (
%)
L91 immunization engender long lasting memory CD4 T cells
L91 immunization eradicate bacteria by
eliciting strong Th1 and Th17 response
L91 immunization generates unique subset
of IFN-γ+IL-17+ polyfunctional Th17 cells
L91 engenders memory generation
Whether immunotherapy with L91
elicits immune response in TB
patients?
Lipidated peptide exhibits T cell proliferation in PBMCs isolated from TB patients and their close contacts
T c
ell p
rolife
rati
on
(S
tim
ula
tio
n i
nd
ex) ****
n= 53 (TB patients)
PBMCs (TB patients) + L91 + IL-2 96h Immune response
9.7±1.5 5.2±0.8
L91 stimulation elicits IFN-γ secretion by the CD4 T cells of TB patients
CD
4+IF
Nγ
+ T
cells (
%)
3.6±0.6
Medium Pam2Cys L91
IFNγ
CD4 *** **
ns
L91 stimulation induces IL-17 secretion by the CD4 T cells of TB patients
CD
4+
IL-1
7+ T
cells (
%)
5.7±1.6 4.9±0.4 IL
-17
CD4
12.5±1.2
Medium Pam2Cys L91
*** **
ns
L91 stimulation generates polyfunctional Th17 cells
IFN
-γ +
IL-1
7A
+ C
D4
T c
ell
s (
%)
***
**
ns
2.8±0.4
(n=14)
3.7±0.6
(n=12)
8.3±1.2
(n=14)
Medium Pam2Cys L91
IFN
-γ
IL-17A
L91 expands the pool of memory T cell
Medium Pam2Cys L91
CD45RA
CD
45R
O
3.8±0.4
(n=28)
4±0.4
(n=15)
7.7±0.8
(n=21)
CD
45
RA
+C
D4
5R
O+
CD
4 T
ce
lls
(%
) ***
**
ns
F91 specific antibodies are absent in TB endemic population
Conclusions
Promiscuous lipidated peptide can elicit robust Th1 and
Th17 response in human PBMCs isolated from TB patients and their closed contacts
L91 immunization induces unique subset of co-expressing CCR6 and CXCR3 Th17 cells, which secrets both IFN-γ and IL-17
No anti-peptide antibodies in population so L91 has
great potential to be a future vaccine candidate for TB endemic area
Immunotherapy with L91 may reduces risk of generation
of drug resistant Mtb
Thanks
Acknowledgment Dr. Javed N Agrewala Professor David C Jackson Professor Ashok K Janmeja CSIR DBT